Genes that are both expressed and required during infection are the best targets for vaccines or antimicrobial therapy, Successful vaccines have been directed against antigens that are expressed during infection and which are essential for the bacteria to cause disease (e.g., cell surface structures, adhesins, toxins, enzymes). In this Project we will use a whole-genome approach to identify essential genes in-vitro, and also those specifically expressed and required in vivo. We will start with Yersinia pestis ("Yp", human strain CO92), for which there exists a complete annotated genome sequence, animal models and vectors for transposon mutagenesis. For CO92 we will construct whole-genome cDNA microarray sequences that contain all predicted open reading frames, and use them to study Yp transcription in vitro and in vivo, and to track transposon mutants during infection. We will use a mini-Tn5 transposon to mutate CO92 to generate a library of mutants, and build a library of 50,000 clones (10x genome coverage), and do microarray transposon tracking (MATT) of insertion sites for 15,000 transposon mutants to identify essential genes. Using the rat bubo model (Project 8: Hinnebusch), we will use wild-type Yp to produce bubos from which we will extract Yp RNA for microarray analysis of gene expression, and compare expression to that of bacteria grown in culture media (at 37C and 26C) to find genes that are upregulated in vivo. We will use pools of transposon mutants (in the rat model, using MATT) to identify which transposon mutants could not colonize or persist in the lesions: this identifies candidate virulence mutants. We will then determine the overlap of genes required in bubonic and pulmonary infections, since the pulmonary mode is the most potentially devastating bioterrorism weapon. We will use MATT to identify genes essential for Yp survival in a mouse pulmonary model. Pools of mutants that have a phenotype in the rat bubo model or mouse pulmonary model will be tested in a primate model of pulmonary infection to identify genes of most importance in human infection. As we progress, we will apply similar techniques to other potential agents of bioterrorism, specifically Francisella tularensis.